Legionella colonization and 3D spatial location within a Pseudomonas biofilm

Biofilms are known to be critical for Legionella settlement in engineered water systems and are often associated with Legionnaire’s Disease events. One of the key features of biofilms is their heterogeneous three-dimensional structure which supports the establishment of microbial interactions and confers protection to microorganisms. This work addresses the impact of Legionella pneumophila colonization of a Pseudomonas fluorescens biofilm, as information about the interactions between Legionella and biofilm structures is scarce. It combines a set of meso- and microscale biofilm analyses (Optical Coherence Tomography, Episcopic Differential Interference Contrast coupled with Epifluorescence Microscopy and Confocal Laser Scanning Microscopy) with PNA-FISH labelled L. pneumophila to tackle the following questions: (a) does the biofilm structure change upon L. pneumophila biofilm colonization?; (b) what happens to L. pneumophila within the biofilm over time and (c) where is L. pneumophila preferentially located within the biofilm? Results showed that P. fluorescens structure did not significantly change upon L. pneumophila colonization, indicating the competitive advantage of the first colonizer. Imaging of PNA-labelled L. pneumophila showed that compared to standard culture recovery it colonized to a greater extent the 3-day-old P. fluorescens biofilms, presumably entering in VBNC state by the end of the experiment. L. pneumophila was mostly located in the bottom regions of the biofilm, which is consistent with the physiological requirements of both bacteria and confers enhanced Legionella protection against external aggressions. The present study provides an expedited methodological approach to address specific systematic laboratory studies concerning the interactions between L. pneumophila and biofilm structure that can provide, in the future, insights for public health Legionella management of water systems.


systems
Legionella pneumophila is a well-known waterborne pathogen responsible for the severe, and often fatal, pneumonia named Legionnaires' Disease 1,2 .L. pneumophila is a very intriguing and complex microorganism which exhibits multiple adaptation and survival mechanisms in the environment, according to the conditions to which it is exposed [2][3][4] .
Protozoa and biofilms are reported as key ecological niches for Legionella settlement and survival in water systems 5 .Protozoa are known to graze the microcolonies of the biofilm, in a prey-predator relationship, and are able to shape the microbial community including the number of pathogens 1,6 .However, the specific role of biofilms in Legionella survival and replication in biofilms is not consensually accepted among researchers 2,4,6 .While some researchers advocate that Legionella growth requires a protozoan host 7,8 , others argue that Legionella is able to colonize and survive in biofilms without intracellular replication 9,10 .Rogers et al. 11 and Wadowsky et al. 12 stated that the presence of non-legionellae bacteria could favor Legionella growth.Later, Surman et al. 9 while using a model water system showed that L. pneumophila was able to proliferate within biofilms without protozoan intracellular replication, as long as other bacterial species were present.More recently, Stewart et al. 13 showed that biofilms composed of Klebsiella pneumoniae and Flavobacterium sp.allowed Legionella persistence for long periods.
Biofilms are complex three-dimensional (3D) heterogeneous structures of microorganisms encased in selfproduced extracellular polymeric substances (EPS) 5,14 .Engineered water systems are complex networks that

Bacterial strains and culture maintenance
The bacterium used to form the biofilms was P. fluorescens ATCC 13525 T .Bacteria were grown overnight at 30 ± 3 °C under agitation in 100 mL of sterile R2 (0.5 g/L peptone, 0.5 g/L glucose, 0.1 g/L magnesium sulphate • 7H 2 O, 0.3 g/L sodium pyruvate, 0.5 g/L yeast extract, 0.5 g/L casein hydrolysate, 0.5 g/L starch soluble and 0.393 g/L di-potassium phosphate•3H 2 O).All components were purchased from Merck (Darmstadt, Germany).
L. pneumophila serogroup 1 (WDCM00107), an environmental isolate, was used throughout this work.The choice relied on the fact that L. pneumophila is responsible for approximately 90% of the reported cases of legionellosis 21 .Bacteria was grown on buffered charcoal-yeast extract (BCYE) agar (Merck, Portugal) at 37 °C for 2 days.

Preparation of the biofilm set-up
In this study, polyvinyl chloride (PVC) coupons placed inside 12-well plates were used to grow biofilms.PVC was selected since it is often found in water engineered systems and past studies showed that it supports biofilms colonized by Legionella 11 .Coupons were sonicated in a 10% sodium dodecyl sulphate (VWR International, Portugal) solution for 5 min.To remove any remaining detergent, coupons were rinsed with tap water and then sonicated again in ultrapure water.Afterwards, the surfaces were rinsed in ultrapure water, air dried, and sterilized with ultraviolet (UV) radiation (254 nm) for 60 min each side.Double-sided adhesive tape was placed in each plate well, sterilized with UV radiation for 60 min, and finally, the sterile coupons were glued in place.

Biofilm formation and Legionella spiking
An overnight culture of P. fluorescens ATCC 13525 T was harvested by centrifugation at 4000 rpm for 10 min at 25 °C (MegaStar 600R, VWR International, Portugal).Cell concentration was adjusted to an optical density (OD 610 nm) of 0.7 in fresh R2, which is equivalent to approximately 10 8 colony-forming units per mL (CFU/mL).
Each well was filled with 3 mL of the prepared bacterial suspension.The plates were then incubated for 14 days at 30 °C under stagnation.Three days after starting biofilm formation, biofilms were spiked with a suspension of L. pneumophila containing 10 9 CFU/mL and incubated again under the same conditions.Culture media was replaced by fresh R2 every 2 days.

Biofilm sampling
Coupons were sampled after 3, 4, 7, 9, 11 and 14 days for biofilm analysis.In the 12-well plates, the bulk media was gently removed and rinsed with sterile saline solution (8.5 g/L) to remove planktonic cells.Coupons were kept in saline solution or let to air dry for imaging (detailed procedures described in "Optical coherence tomography (OCT)" and "Peptide nucleic acid (PNA) -Fluorescence in situ hybridization (FISH)" sections).For quantification of the sessile cells in the biofilms, coupons were gently removed from the 12-well plates, and were transferred to 15 mL centrifuge tubes (VWR, Portugal), containing 2 mL of saline solution.To disaggregate the biofilms and resuspend the cells, the tubes were submitted to three alternate cycles of 30 s sonication (Ultrasonic Cleaner USC-T, 45 kHz, VWR International, Portugal), followed by 30 s of vortexing. www.nature.com/scientificreports/

Biofilm analysis
Optical coherence tomography (OCT) Biofilms were imaged as described by Silva et al. 29 , directly from the 12-well plates with sterile saline solution, using spectral-domain Optical Coherence Tomography (OCT; Thorlabs Ganymede, Thorlabs GmbH, Germany) with a central wavelength of 930 nm 29 .The captured volume was 2.49 × 2.13 × 1.52 mm (y × z × x), consisting of 509 × 313 × 1024 pixels 3 .For each coupon, 2D and 3D imaging were performed with a minimum of five and three different fields of view (FoV), respectively.The acquired OCT images were processed with the software Biofilm Imaging and Structure Classification Automatic Processor (BISCAP) 31 , available at https:// github.com/ diogo narci so/ BISCAP.In brief, for each 2D-OCT image, the pixels at the substratum were identified, and a threshold for the pixel intensity was calculated, enabling binarization of pixels as biomass or background, thereby distinguishing the biofilm from the liquid bulk phase 32 .The 2D image processing was extended to the 3D-OCT images, which correspond to 509 2D-OCT images as described by Narciso et al. 31 .BISCAP software was used to quantify the biofilm average thickness, compaction parameter and porosity.The specific definitions of the average thickness, compaction parameter and porosity can be found in Narciso et al. 31,32 .Briefly, the average thickness refers to the total length between the bottom and top of the biofilm.The compaction parameter, proposed by Narciso et al. 32 , measures the compactness of the biofilm; it represents the ratio between the continuous biomass pixels to the total number of pixels (biomass + water) between the bottom and top interfaces.The delivered values range from 0 to 1, where values closer to 1 correspond to very compact biofilms (with low empty spaces).The porosity was defined as the fraction of background voxels in the biofilm region, and varies between 0 and 1, as proposed by Narciso et al. 31 .

Peptide nucleic acid (PNA)-fluorescence in situ hybridization (FISH)
To track the spatial position of L. pneumophila inside biofilms, the PNA probe PLPNE620 (5′-CTG ACC GTC CCA GGT-3′) (Cambridge Research Biochemicals United Kingdom) was used, since it was successfully applied to detect the pathogen in past studies 28 .After rinsing with saline solution, coupons were allowed to air dry at room temperature.The PNA hybridization and washing step were performed according to Wilks et al. 28 .Control experiments were carried at each sampling timepoint to ensure that no cross-staining between P. fluorescens and L. pneumophila occurred, nor EPS staining.For that, control biofilms of P. fluorescens were hybridized with the PNA probe in the same conditions formerly described.

Episcopic differential interference contrast (EDIC)/epifluorescence (EF) microscopy
The stained coupons were examined using a Nikon Eclipse CFI60 episcopic differential interference contrast (EDIC) coupled with epifluorescence (EF) microscope, using a 50 × Plan APO objective (Best Scientific, UK).The EDIC channel was used to visualize the microscale structure of biofilms, while the TRITC channel was used to visualize and track the red labelled L. pneumophila.Representative images were taken over 20 fields of view and processed using ImagePro image capture software.The images were obtained with equal exposure times and gain values.

Confocal laser scanning microscopy (CLSM)
The stained coupons were also observed with a white light laser (WLL) at excitation wavelength of 565 nm and a 405-diode laser at excitation wavelength of 398 nm, using a 40 × glycerol objective lens in a Leica STELLARIS (Leica Stellaris, Leica Microsystems, Germany).A minimum of six stacks of horizontal plane images (512 × 512 pixels, corresponding to 387.5 × 387.5 µm) with a z-step of 0.36 µm were acquired for each sample.IMARIS 9.1 software (Bitplane, Switzerland) was used to create 3D projections of biofilm structures.The plugin COMSTAT2 from ImageJ was used to quantify the biovolume (µm 3 /µm 2 ) 33 .The biovolume was defined as the overall volume of cells (µm 3 ) divided by the substratum area, and it can be used to estimate how much biomass is in a biofilm 33 .

Quantification of sessile cells
To assess P. fluorescens culturability, serial dilutions were performed and plated in triplicate in plate count agar (PCA) (Oxoid, Portugal).Plates were incubated at 30 °C for 24 h for colony-forming units (CFU) enumeration.After assessing P. fluorescens culturability, biofilm suspensions were thermal treated (50 °C for 30 min) to eliminate P. fluorescens from the sample.The treated suspensions were spread onto the selective media BCYE-GVPC (buffered charcoal yeast extract supplemented with glycine, vancomycin, polymyxin and cycloheximide) agar and incubated at 37 °C up to 10 days to assess Legionella culturability.

L. pneumophila migration within the biofilm during the initial 24 h
The migration of L. pneumophila within the biofilm was followed over time during the first 24 h after spiking.Biofilm was sampled, labelled with the 16S rRNA PNA probe and imaged using CLSM, according to the previously described methods ("Peptide nucleic acid (PNA)-fluorescence in situ hybridization (FISH)" and "Confocal laser scanning microscopy (CLSM)" sections).The biofilms were analysed at 5 min, 15 min, 30 min, 2 h, 4 h, 6 h, 10 h, 20 h and 24 h after Legionella spiking.

Statistical analysis
The experimental data were analysed using the software GraphPad Prism 9.0 for Windows (GraphPad Software, USA).Three independent experiments were performed.The mean and standard deviation (SD) for each set of results were calculated.Results were compared using an ANOVA single-factor statistical analysis and Student's t-test.The level of significance was set for p-values < 0.05.www.nature.com/scientificreports/

Results
P. fluorescens and L. pneumophila culturability P. fluorescens culturability per volume of biofilm did not show statistically significant differences over time between the control biofilm (P.fluorescens alone-Pf) and those spiked at day 3 with L. pneumophila (Pf + Lp)-Fig.1a.In both cases, the amount of P. fluorescens (~ 9 log 10 CFU/cm 3 ) did not significantly change between days 3 and 14 (p > 0.05).On the other hand, L. pneumophila was recovered for 11 days from the mixed biofilm of Pseudomonas and Legionella, but as shown in Fig. 1b, the culturable numbers of L. pneumophila per biofilm volume had 1-log reduction (p < 0.0001) between days 4 and 7 and maintained around 5 log 10 CFU/cm 3 until the end of each experiment.This reinforces the notion that L. pneumophila is able to colonize and persist (at least for 11 days) in P. fluorescens biofilms, confirming the previous work from Stewart et al. 13 .

Biofilm mesoscale structure
The mesoscale structures of the control biofilms of P. fluorescens (Pf-without L. pneumophila) were compared with those spiked with L. pneumophila (Pf + Lp) on day 3. Figure 2 depicts representative 2D-OCT biofilm images for both conditions (Pf and Pf + Lp biofilms).
When analyzing the control P. fluorescens biofilm mesoscale structure over time, it can be seen that the regular and flat structure observed on day 3 (Fig. 2A) is similar to the one found on day 4 (Fig. 2B).Over time, P. fluorescens control biofilms (Fig. 2C and D) tend to become more irregular and exhibit more empty spaces (colored in blue).A similar behavior is observed for the P. fluorescens biofilms spiked with L. pneumophila, except that, for longer incubation periods, the spiked biofilms (Fig. 2F and G) tend to be significantly thicker than the control biofilms, and show increased empty channels.Not surprisingly, the area occupied by the empty channels is more pronounced in the top of the biofilm than in the bottom, for the control and spiked biofilms.
No significant changes were observed in the thickness profile of the P. fluorescens control biofilms (Fig. 3a, green bars) which was found to be 61 ± 11 µm over the 14 days experimental period.The other mesoscale parameters showed significant changes from days 3 to 4 (p < 0.05): while porosity (Fig. 3b) increased, the compactness of the biofilm has been reduced (Fig. 3c).From day 4 until the end of the experiment, the above mentioned parameters remained stable, suggesting the biofilm structure reached the plateau 34 .
Upon L. pneumophila spiking to the P. fluorescens biofilms (Pf + Lp), no significant changes in thickness were noticeable between days 3 and 4, as shown in Fig. 3a (orange bars).However, from days 7 to 14, biofilms with L. pneumophila became significantly thicker than the ones of P. fluorescens alone (p < 0.0001), reaching the highest thickness of 90 µm by day 11.The porosity and compactness did not change (p > 0.05) between days 3 and 4 (Pf + Lp, orange bars).Changes were only noticeable later, by day 7, as the porosity increased (p < 0.05) and the compaction decreased (p < 0.05), to values like the ones from the non-spiked biofilms (Pf).

Legionella spatial location
To study the spatial location of L. pneumophila within the P. fluorescens biofilms, the microscale structure of the spiked biofilms was characterized by episcopic differential interference contrast microscopy (EDIC) with epifluorescence (EF) and by Confocal Laser Scanning Microscopy (CLSM).L. pneumophila is labelled red through the specific 16S rRNA PNA probe (PLPNE620).Representative images of P. fluorescens biofilms stained with the same PNA probe and visualized at the EDIC/EF (Fig. S1) and CLSM (Fig. S2) are provided in the Supplementary Information.These images show that there is no cross-staining between the bacteria nor any interaction with the biofilm EPS (no red signal is observed).Figures 4 and 5 show representative EDIC/EF and CLSM images of the spiked biofilms, respectively, and show that L. pneumophila was widespread within the coupons, and also emphasize the success of bacteria in colonizing the P. fluorescens biofilm.
The EDIC/EF microscopy images allowed to qualitatively characterize the biofilm microscale structure and to visualize the predominant location of L. pneumophila within it.Direct observation of biofilms 24 h after the L. pneumophila (day 4) spiking, EDIC/EF imaging revealed the presence of microcolonies (Fig. 4-white arrows) and the diffuse fluorescence surrounding them is indicative of eDNA in the accumulating EPS.The presence of microcolonies was further confirmed with the OCT since each of the individual black dots are too large to be individual bacteria and more likely to be microcolonies (approximately 10-20 microns in diameter).In general, from days 4 to 14, there were some highly colonized areas (Fig. 4-yellow arrows) separated by others with less biofilm density, showing the heterogeneous nature of biofilms.Biofilms showed increased thickness with time, which is particularly noticeable by day 14 (Fig. 4E).In this figure, biofilm microcolonies seem to be brighter and more well-defined than in previous days, which reflects the growth of the microcolonies and the expected higher rRNA content present in the biofilm.
L. pneumophila red fluorescing cells can also be seen (under the TRITC filter), evidencing its widespread distribution within the biofilm.Regions, where the coupon was scratched or with some more prominent biofilm aggregates, had massive L. pneumophila clumps.Some water channels were also observed in the biofilm, but no significant amounts of L. pneumophila were observed near such water channels.The intensity of the red fluorescing cells (Fig. 4B, D and F) seems to become faint over time (particularly by day 14).
The detailed investigation of the L. pneumophila spatial position within the P. fluorescens biofilm was established via confocal imaging.The three-dimensional reconstructions of the biofilms-Fig.5-revealed the presence of P. fluorescens (observed as green due to the autofluorescence conferred by self-produced pigments 35,36 ) and L. pneumophila in very similar proportions.Furthermore, L. pneumophila was mostly located in the bottom layers of the biofilm.This was observed for the whole experimental period.

L. pneumophila migration within the biofilm during the initial 24 h
The migration of L. pneumophila within the P. fluorescens biofilm was monitored over a 24 h period after L. pneumophila spiking, using confocal imaging (Fig. 7).No L. pneumophila was observed in the 5 initial minutes after the spiking.A thin layer of L. pneumophila was detected on the top surface of the biofilm 15 min after spiking.Over time, an increase in L. pneumophila on the top of the biofilm was observed, suggesting an accumulation of the bacteria.By the 4 h mark, a significant amount of L. pneumophila started to appear in the bottom layers of the biofilm, simultaneously with a bacterial decrease on the top.This migration continued progressively, with L. pneumophila becoming predominantly located at the bottom of the biofilm by the end of the 24 h observation period.

Discussion
L. pneumophila entrance in the 3-days P. fluorescens biofilm was evaluated regarding the impact on the biofilm structure and on the bacteria positioning over 11 days.

L. pneumophila colonization of the P. fluorescens biofilm-impact on the biofilm structure
When L. pneumophila colonizes the P. fluorescens biofilms, they maintained their mesoscale structure (quantified through thickness, porosity, and compaction parameter of the 3D-OCT images) as no significant differences were found between days 3 and 4 (before and 24 h after L. pneumophila spiking, respectively)-Fig.3 (orange bars).Differences in the Legionella spiked biofilms structure were only noticeable later (when sampling the biofilm at day 7), as they tended to rearrange into similar characteristics as those from the control (P.fluorescens alone) biofilms.Lee et al. 37 reported a delay in biofilm development, concluding that the development of mixed-species is slower (1-or 2-day delay) than single-species biofilms.The control biofilm (P.fluorescens only) rearranged structurally between days 3 and 4 (Fig. 3, green bars), and then remained stable, suggesting that the biofilm development reached its plateau by day 4.However, thickness followed a different trend: from days 7 to 14, the spiked biofilms became progressively thicker (~ 30%) than the Pf controls (Fig. 3a, green bars).A similar behaviour was found by Koh et al. 38 who described that the thickness of P. aeruginosa biofilms exposed to a waterborne pathogen, Cryptosporidium parvum, increased when compared to the control biofilms.Also, Puga and colleagues 39 reported that spiking Listeria monocytogenes to pre-established P. fluorescens biofilms led to an EPS matrix over-production.According to other authors, mixed-species biofilms might have an increased biomass production 37,40 , which can be related to events of space optimization due to different bacterial interactions 41 .
The other mesoscale characteristics of the biofilms (including porosity and compaction parameter) suggest that regardless of the Legionella presence, the dominant biofilm structure is the one from the P. fluorescens-the first colonizer.In addition, the present results show that the cell density of P. fluorescens (Fig. 1a) was not significantly affected by the presence of L. pneumophila.Pang et al. 42 while studying the colonization of P. fluorescens biofilms by L. monocytogenes also concluded that P. fluorescens cell density did not change with the presence of L. monocytogenes.
The observed dominance of P. fluorescens over L. pneumophila in the biofilm may be related with the fact that P. fluorescens is a well-known EPS producer strain 20,42,43 .It has been previously reported that microorganism producers of EPS have competitive advantages over other bacteria if they are the first colonizers 44 .Some authors argue that Legionella is able to form biofilms on its own under very well-defined laboratory conditions 45,46 , but with no significant amounts of EPS 46 .However, under real environmental scenarios, Legionella colonizes preestablished biofilms, as a secondary colonizer 5 .Furthermore, the large amounts of EPS produced by P. fluorescens might enhance the physical fixation/entrapment of L. pneumophila and will allow the establishment of more robust biofilms with increased cohesion 39,47 , arguably more difficult to suffer slough-off.

L. pneumophila location within the P. fluorescens biofilm
Results showed that L. pneumophila successfully colonized and persisted in a P. fluorescens biofilm at least for 11 days.Representative CLSM images of 4-, 7-and 14-days biofilms spiked with L. pneumophila; The latter was stained with a PNA probe (in red).The confocal images are 3D projections obtained using IMARIS, and the white scale bars are 50 µm.A representative image of the control P. fluorescens biofilm stained with the PNA probe is provided in Supplementary Information (Fig. S2).www.nature.com/scientificreports/ The EDIC images showed that PNA-L.pneumophila signal became faint over time, which seems to be consistent with the Legionella biovolume (Fig. 6) and culturability (Fig. 1b) decrease over time.In all situations this might be a consequence of L. pneumophila entering a non-culturable but viable state (VBNC).It is reported that www.nature.com/scientificreports/VBNC cells have lower metabolic activity and lower levels of rRNA 48,49 .If the amount of rRNA decreases, and since the PNA probe binds specifically to 16S rRNA molecules, one might expect that the intensity of the signal (observed as a red color) will also decrease 48,50 .Former studies showed that there are a vast number of 16S rRNA molecules per bacterium compared to copies of the gene 51,52 .Thus, the bright and further decrease in the PNA-FISH signal is arguably due to decreasing 16S rRNA content and not from the very low number of copies of the 16S rRNA chromosomal gene.The ability of Legionella to enter into the VBNC state has been demonstrated by several authors [53][54][55] .Gião et al. 56 and Alleron et al. 57 induced L. pneumophila cells into VBNC state through chlorine and monochloramine exposure, respectively.Indeed, the former remained infective in an Acanthamoeba animal model.Other studies also concluded that under a low nutrient environment, Legionella would lose its culturability 58 , and that VBNC cells exhibit smaller cell sizes 59,60 .An alternative explanation for the faint signal might be that, over time, L. pneumophila is washed-off of the biofilm, as the medium is replaced every 2 days.
Regarding the spatial positioning of the bacteria, the CLSM images (Fig. 5) show that bacteria were essentially positioned in two distinct layers.While L. pneumophila was positioned in the bottom of the biofilm, P. fluorescens was located in the upper layers (Fig. 5).Two distinctive physiological aspects between both bacteria are related to the oxygen consumption and nutrients uptake.While P. fluorescens metabolizes carbon sources and is aerophilic 61 , L. pneumophila has very specific nutritional requirements and behaves as a microaerophilic microorganism 62 , thus growing in the presence of oxygen but better at lower oxygen levels.Since the transport of nutrients and oxygen is higher at the biofilm top interface 63 , the relative positioning of Pseudomonas and Legionella inside the biofilm is a win-win situation for both bacterial species.This also explains why L. pneumophila is not placed around water channels (observed in the EDIC/EF imaging-Fig.4), as the primarily function of water channels is to favor mass transport (nutrients, oxygen, waste-products, etc.) between the biofilm and the surrounding liquid 64 .And expectedly higher oxygen and nutrients concentrations might be found on those areas 11 .It is not surprising though that Legionella is located at the bottom layers of the biofilm where micro-environments with lower oxygen levels can be found.Additionally, it has been demonstrated that the EPS producer cells and their descendants (in the case of the present study-P.fluorescens) will be positioned in the biofilm top layers, keeping privileged access to nutrients and oxygen and allowing such bacteria to dominate the biofilm 65 .Indeed, the OCT imaging (Fig. 2) demonstrated that most of the empty spaces-that are linked to events of mass transfer-are located in the upper layers of the biofilm 66 .This also supports the former conclusions of the present work that by the middle of the experimental biofilms colonized by L. pneumophila presents the same mesoscale structure properties (except for thickness) as the one from the P. fluorescens control biofilm.
Finally, from the Legionella perspective, being at the bottom of the biofilm (the EDIC/EF imaging showed that many cells were in the scratches of substratum material), Legionella will be more protected than in the top layers against external harshness like biocides or thermal shocks.There are several studies demonstrating the ability of P. fluorescens biofilms to shield pathogens 39,67,68 .

How long does L. pneumophila need to reach the bottom of the P. fluorescens biofilm?
The time-lapse representative CLSM images of L. pneumophila colonization of the pre-established P. fluorescens biofilm over the initial 24 h after L. pneumophila spiking (Fig. 7) show that L. pneumophila starts to adhere, to a greater extent, to the top of the biofilm within 15 min after spiking.It is somehow surprising that no L. pneumophila was observed in the first 5 min, as the experiment was conducted under stagnation (no flow) conditions.Former work demonstrated that sedimentation significantly affects bacterial attachment and mass transfer, even under low flow conditions 69,70 .Under no-flow conditions, the sedimentation effect is even higher, and the entire biofilm was surrounded by Legionella.Therefore, the fact that L. pneumophila took between 5 and 15 min to adhere to the top layer of the P. fluorescens (Fig. 7, Top, 15 min), is likely due to the multiple adaptation strategies that Legionella can undergo.Several studies show that the morphological changes of Legionella appendages are critical to the interactions within host-protozoa and allow the bacteria to switch between the replicative and transmissive phases 71 .The study from Abdel-Nour et al. 72 also shows that adhesins, in particular, collagen-like adhesin is important for Legionella attachment to surface, biofilm formation and auto-aggregation.
Once L. pneumophila interacts with the top layer of the biofilm it quickly (between 2 and 4 h) reaches the bottom of the P. fluorescens biofilm.Considering that the pre-established 3 days biofilm have an average thickness of ~ 58 µm, the average linear migration speed of L. pneumophila across the biofilm is ~ 22 µm/ h.This migration speed is consistent with the range proposed by Picioreanu et al. 73 for the computational model simulation of P. aeruginosa biofilm formation, which accounted with many factors, including cells motility and twitching motility.Albeit it is important to remark that in the present study, L. pneumophila was not the first colonizer and already encountered a pre-established thick biofilm, with high cellular density (~9 log 10 CFU/cm 3 ) and a very well organized mesoscale structure (Fig. 2A), which could have been a constraint to L. pneumophila and migration.Puga et al. 39 attributed the differences between the colonization of 48 h pre-established P. fluorescens biofilms by L. monocytogenes formed under different conditions to the physical impediment bacteria face when entering different structures of the already established biofilms.It seems that apart from the hypotheses already discussed regarding the distinctive physiological aspects between the two bacteria species (nutrient and oxygen requirements), L. pneumophila might had also taken advantage of the empty spaces found in the P. fluorescens biofilm (Fig. 2A-colored in blue) to quickly move across the biofilm and reach its bottom.As previously discussed, no significant changes were observed at the mesoscale structure of the biofilm (Figs. 2 and 3) reinforcing the idea that L. pneumophila took advantage of the already existing biofilm structure rather than creating transient biofilm structures (like pores or channels) as reported in other works 74 .
After 4 h, the significant decrease of L. pneumophila in the top layer of the biofilm is arguably related to sedimentation and with the fact that L. pneumophila keeps moving across the biofilm since a significant increase of red stained L. pneumophila cells is observed in the bottom of the biofilm.Between 20 and 24 h all the L. www.nature.com/scientificreports/pneumophila is positioned in the bottom layer of the P. fluorescens biofilm (Fig. 7), in a very high concentration (~7 log 10 CFU/cm 3 , Fig. 1b).The 24 h L. pneumophila concentration in the biofilm and in the bulk (~8 log 10 CFU/ mL), raises the question of whether L. pneumophila is or not able to replicate within a mono-specie biofilm even if it is over a small timespan.A proper answer to this question requires further investigation.Of note is that the L. pneumophila numbers provided were obtained by culturability, thus likely reflecting an underestimation the true amounts of bacteria in the system.The present work brings new insights for the discussion about Legionella and biofilms interactions concerning the structural changes and relative location of L. pneumophila within the P. fluorescens biofilm.Although the experimental design does not aim to mimic the interactions of biofilm-Legionella in engineered water systems, it provides an expedite approach to tackle some fundamental questions regarding such interactions.The combination of micro and mesoscale techniques provided significant and complementary information that can be used in future works and in real studies.In this scope, it worth to highlight that OCT imaging showed to be a powerful non-staining technique that rapidly describes the biofilm 3D meso-scale structure, microcolonies accumulation and water filled areas.
It is important to remark that the results obtained in the present study might be different concerning the pre-established biofilm species used or the Legionella species/strains considered or the introduction of host cells.
Finally, the proposed experimental model offers to the scientific community a platform to study, in a systematic way, several questions related to mechanistic and physiological aspects of Legionella-biofilms interactions, including transmission, the behaviour of mutants (among many others) which might allow, in the future, to better understand the bacteria dynamics in the complexity and variability of real systems.
Future work is focused on answering to some of the questions raised during this study regarding whether L. pneumophila replicates or not in the biofilm and whether it enters VBNC states or wash-off from the biofilm over time.Since biofilm detachment is critical from a public health perspective of legionellosis prevention the model will also be revised to consider this aspect in future works.

Figure 1 .
Figure 1.Bacteria culturability expressed per volume of biofilm (log 10 CFU/cm 3 ) (a) P. fluorescens and (b) L. pneumophila recovered from biofilm over time.The mean ± standard deviation is shown.Statistically significant differences are represented for p < 0.0001 by ****; ns: not statistically significant.

Figure 3 .
Figure 3. Thickness (a), porosity (b) and compaction parameter (c) of the control (Pf) -green bars and spiked (Pf + Lp) -orange bars biofilms over 14 days.Values were extracted from 3D-OCT images with the BISCAP software.The mean ± standard deviation is shown.Statistically significant differences are represented for p < 0.05 by *, < 0.01 by **, < 0.0005 by *** and < 0.0001 by ****.Error bars in black, green and orange refer to significant differences between control and spiked biofilms, between the control biofilms and between the spiked biofilms, respectively.L. pneumophila spiking is indicated by an arrow.

Figure 4 .
Figure 4. Representative EDIC/EF images of 4-, 7-and 14-days biofilms spiked with L. pneumophila; the latter were stained with a PNA probe (in red).Biofilms were visualized using the EDIC channel (images A, C and E) and using a TRITC filter for fluorescence (images B, D, and F).White arrows indicate microcolonies and yellow arrows indicate areas highly colonized.Bars represent 10 µm.Magnification × 500.A representative image of the control of P. fluorescens biofilm stained with the PNA probe is provided in Supplementary Information (Fig. S1).

Figure 5 .
Figure 5.Representative CLSM images of 4-, 7-and 14-days biofilms spiked with L. pneumophila; The latter was stained with a PNA probe (in red).The confocal images are 3D projections obtained using IMARIS, and the white scale bars are 50 µm.A representative image of the control P. fluorescens biofilm stained with the PNA probe is provided in Supplementary Information (Fig.S2).

Figure 6 .
Figure 6.Biovolume of P. fluorescens and L. pneumophila in spiked biofilms (Pf + Lp) developed under 14 days.Values were extracted from confocal images with the COMSTAT plugin.The means ± standard deviations are shown.Statistically significant differences are represented for p < 0.0001 by ****.
Biofilms are a key ecological niche for Legionella persistence in water systems, although the microbial interactions between them are still poorly understood.The laboratory model developed in this study deciphered some of the interactions of L. pneumophila and P. fluorescens biofilms.The main findings of this work are: (a) the overall dominant biofilm structure is the one provided by P. fluorescens, regardless of the L. pneumophila colonization; (b) the spiked biofilms are thicker than the ones from P. fluorescens alone; (c) L. pneumophila reaches in 2-4 h the bottom of the biofilm, were it is preferentially positioned over the 11 days of the trial, thus being more protected from external stressors, and (d) both PNA-labelling and L. pneumophila culturability suggest that by the end of the experiment Legionella might be entering a VBNC state for stress survival.